Use of jaz5a for improving drought-resistance in a plant

ABSTRACT

The present invention provides use of a plant gene JAZ5a for improving drought-resistance of a plant. It further provides a method for improving the drought-resistance of a plant, comprising enhancing the expression or activity of Jaz5A in said plant.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a Divisional Application of U.S. application Ser.No. 14/131,184, filed Mar. 24, 2014, which is the National Phase ofInternational Patent Application No. PCT/NL2012/050481, filed Jul. 5,2012, published on Jan. 10, 2013 as WO 2013/006058 A1, which claimspriority to U.S. Provisional Application No. 61/505,391, filed Jul. 7,2011. The contents of which are herein incorporated by reference intheir entirety.

The instant application contains a Sequence Listing which has beensubmitted in ASCII format via EFS-WEB and is hereby incorporated byreference in its entirety. Said ASCII copy, created on Jun. 19, 2017, isnamed 085342-1800Sequence_Listing.txt and is 12 KB.

TECHNICAL FIELD

The present invention belongs to the fields of biotechnology and botany.The present invention relates to a new method for improving droughtresistance of a plant. The invention involves the use of a protein insaid plant for improving drought resistance. The present inventionrelates to the enhancement of the expression or activity of the protein,thereby providing improved drought resistance to a plant in comparisonto a plant not modified to enhance expression of the protein.

BACKGROUND ART

Cabbages mainly include Brassica campestris L. ssp. Pekinensis andBrassica campestris L. ssp. chinensis. Brassica campestris L. ssp.chinensis is also named as green cabbage, and baby Brassica campestrisL. ssp. chinensis in the north of China. Brassica 25 campestris L. ssp.chinensis exhibits high adaptability, growth, productivity andnutrition. It is the most consumed vegetable among various vegetablesand widely grew in the provinces in the regions of Changjiang valley inChina. There are various types and varieties of Brassica campestris L.ssp. chinensis. Cabbages have a short growth period, wide adaptability,and high productivity. They are also easy to plant, which allows for asustained perennial supply. The products of Brassica campestris L. ssp.chinensis are fresh and tender, have rich nutrition and win favor ofconsumers. Brassica campestris L. ssp. chinensis comprises about 30-40%of the total domestic vegetable productivity a year, and also makes asignificant contribution in supplementing vegetables in slack seasonsand balancing the vegetable supply over a whole year. Both the Brassicacampestris L. ssp. Pekinensis and Brassica campestris L. ssp. chinensisfavor cool weather and can be planted perennially. The most suitablegrowth temperature is 15-20° C. In recent years, to meet the marketdemand, cabbages are mainly planted by the technique of intensiveculture. To ensure an even production and supply among the four seasons,Brassica campestris L. ssp. chinensis generally needs to be planted indifferent manners in different seasons. In the past, Brassica campestrisL. ssp. chinensis was mainly planted in spring and winter. Now peoplebegin to plant Brassica campestris L. ssp. chinensis in torrid summerand autumn by various culture manners. This will undoubtedly makeBrassica campestris L. ssp. chinensis subject to the stress from droughtduring its growth, especially in late spring, summer and early autumn.

The Brassica campestris L. ssp. chinensis cultured in the seasons ofhigh temperature can go to the market in bulk after a 20-day culture.However, the high temperatures usually lead to an elongated internode,slowed growth, bitter taste and undesirably increased fiber, etc. Thiswill result in low productivity and poor quality. As a result, the pricerises and the supply falls short of demand. The consumer demand cannotbe met. Brassica campestris L. ssp. Pekinensis has poor tolerance todrought. It is highly drought sensitive in the rosette stage and theheading stage. If the average temperature is too high, the heart leafcan not amplexate to build a tight bulb, or can not bulb up at all. Evenif it constrainedly bulbs up, the heading is loose. In the natural fieldconditions in summer, the production relies on the drought-resistanceplants' capability of forming a normal leafy head. And the capability ofheading formation under the natural high temperature in fields becomesan indication of a drought-resistance in Brassica campestris L. ssp.Pekinensis.

Both the Brassica campestris L. ssp. Pekinensis and the Brassicacampestris L. ssp. chinensis were originally planted in China. Inforeign countries, there is few studies on breeding of cabbages.Varieties of Japanese, Korean and Formosan origins are poor in droughtresistance, and unsuitable for planting in China. Domestically dominantare mainly the disease resistant varieties planted in autumn. Vegetablesof cabbages have a narrow gene library for drought-resistance. Breedingof drought-resistance cabbage variety is limited to the screening amongthe cabbage materials, whereby only some varieties with poor droughtresistance and low stress resistance have been obtained.

To solve these problems, the domestic breeding experts have utilized thetraditional breeding methods to widely screen and culturedrought-resistance varieties of vegetables of cabbages, to introducedrought-resistance genes, and broaden the sources of exploitation, whichimproved the drought-resistant ability of vegetables of cabbages to acertain degree and have produced effect in actual production. However,the current methods are limited to the assessment of drought resistanceunder the local climate and the morphological changes under a hightemperature stress. These methods are not suitable for the temperateareas, which can not provide the field conditions with suitableselection stresses. Even if a single drought-resistance plant wasselected, a series of complicated methods and means would be required tomaintain the drought-resistance in the seeds collected until the nextspring. The screening requires a long period, and is geographicallylimited, which can not provide a drought resistant variety universallyadaptable. Therefore, it is an urgent task in breeding ofdrought-resistance vegetables of cabbages to intensively study theoccurrence and development of the drought damages during the seedlingstage, and to develop a method and technique for screening droughtresistance in seedling stage, which provides improved operability,stability, efficiency and adaptability. The traits closely associatedwith the drought resistance in cabbages are of a quantitative nature,which poses great difficulties in genotyping. Particularly for molecularbreeding, the difficulties include not only the limited number of DNAmarkers useful in the auxiliary selection, but also the inconsistence ofthe number and the significance of the quantitative traits loci (QTL).Therefore, since the genome sequencing of cabbages is not finished yet,and the study on functional genome study is gaining increasinginterests, there is a need for a quick, sensitive and efficientqualitative analysis on the various traits in plant and the DNAprofiles, and a quantitative analysis on the phenotypes in plant andchanges in gene expressions, which is usefully in the breeding ofdrought-resistance cabbages.

There is a need in the art for identifying plant drought-resistancegenes.

SUMMARY OF THE INVENTION

It is an objective of the current invention to provide for droughtresistance in a plant. With plants provided with drought resistance, orplants with improved drought resistance it is e.g. possible to obtainhigher yields of crop and/or plant product when the plant is subjectedto a period or periods of drought when compared to plants not providedwith (improved) drought resistance. It was found a plant can be providedwith (improved) drought resistance when the expression in said plant ofa JAZ5a gene is enhanced. The current invention thus provides for usesof the JAZ5a gene for providing (improved) drought resistance. Otheraspects of the present invention will be apparent to the skilled personbased on the contents disclosed herein.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows wilting symptoms 15 DOD and onwards, plants were dailygiven a score between 0 and 4 (y-axis) based on which wilting symptomsthey exhibited. Wilting symptoms were expressed as 0, no symptoms; 1,very mild loss of turgor; 2, loss of turgor; 3, severe loss of turgor;4, putatively dead. Red asterisk indicate statistical differences inwilting score between mutant plants and wildtype plants (student'st-test; a<0.05). Black asterisk in legend indicate hetorozygosity of theline. Each graph represents an individual tray.

FIG. 2 shows a representative effect of rehydration one week afterrehydration at 19 DOD or 20 DOD, comparing wild-type plants with35:BcpJAZ5a plants. Clearly, the 35:BcpJAZ5a plants perform better thanthe wild-type plants.

DEFINITIONS

In the following description and examples, a number of terms are used.In order to provide a clear and consistent understanding of thespecification and claims, including the scope to be given to such terms,the following definitions are provided. Unless otherwise defined herein,all technical and scientific terms used have the same meaning ascommonly understood by one of ordinary skill in the art to which thisinvention belongs. The disclosures of all publications, patentapplications, patents and other references are incorporated herein intheir entirety by reference.

Methods of carrying out the conventional techniques used in methods ofthe invention will be evident to the skilled worker. The practice ofconventional techniques in molecular biology, biochemistry,computational chemistry, cell culture, recombinant DNA, bioinformatics,genomics, sequencing and related fields are well-known to those of skillin the art and are discussed, for example, in the following literaturereferences: Sambrook et al., Molecular Cloning. A Laboratory Manual, 2ndEdition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.,1989; Ausubel et al., Current Protocols in Molecular Biology, John Wiley& Sons, New York, 1987 and periodic updates; and the series Methods inEnzymology, Academic Press, San Diego.

In this document and in its claims, the verb “to comprise” and itsconjugations is used in its non-limiting sense to mean that itemsfollowing the word are included, but items not specifically mentionedare not excluded. As used herein, the term “comprising”, “having” or“containing” includes “comprising”, “consisting substantively of”,“consisting essentially of”, and “consisting of”. The “consistingsubstantively of”, “consisting essentially of” and “consisting of” arespecific concepts of the generic terms “comprising”, “having” and“containing”.

As used herein, the singular forms “a,” “an” and “the” include pluralreferents unless the context clearly dictates otherwise. For example, amethod for isolating “a” DNA molecule, as used above, includes isolatinga plurality of molecules (e.g. 10's, 100's, 1000's, 10's of thousands,100's of thousands, millions, or more molecules).

The term “polynucleotide”, “nucleic acid molecule” or “nucleic acidsequence” refers to a DNA or RNA molecule in single or double strandedform, particularly a DNA encoding a protein according to the invention.An “isolated nucleic acid sequence” refers to a nucleic acid sequencewhich is no longer in the natural environment from which it wasisolated, e.g. the nucleic acid sequence in a bacterial host cell or inthe plant nuclear or plastid genome. For example, a polynucleotide and apolypeptide in a natural state in the living cell is not isolated orpurified. However, when the same polynucleotide or polypeptide isseparated from the other substances with which it coexist in the saidnatural state, it is called “isolated” and/or “purified”.

Aligning and alignment: Wth the term “aligning” and “alignment” is meantthe comparison of two or more nucleotide sequences based on the presenceof short or long stretches of identical or similar nucleotides. Severalmethods for alignment of nucleotide sequences are known in the art, aswill be further explained below.

“Expression of a gene” refers to the process wherein a DNA region, whichis operably linked to appropriate regulatory regions, particularly apromoter, is transcribed into an RNA, which is biologically active, e.g.which is capable of being translated into a biologically active proteinor peptide or active peptide fragment. An active protein in certainembodiments refers to a protein being constitutively active. The codingsequence is preferably in sense-orientation and encodes a desired,biologically active protein or peptide, or an active peptide fragment.

“Functional”, in relation to proteins (or variants, such as orthologs ormutants, and fragments), refers to the capability of a gene and/orencoded protein to have an effect on a quantitative and/or qualitativefeature(s) of a plant. By modifying the expression level of the gene(e.g. by enhancing expression or reducing expression) the quantitativeand/or qualitative feature of a plant is affected. For example, when aprotein has a function in drought resistance, enhancing gene expressionmay lead to drought resistance. The skilled person will have nodifficulties in testing functionality with regard to abiotic stressessuch as drought.

The term “gene” means a DNA sequence comprising a region (transcribedregion), which is transcribed into an RNA molecule (e.g. an mRNA) in acell, operably linked to suitable regulatory regions (e.g. a promoter).A gene may thus comprise several operably linked sequences, such as apromoter, a 5′ leader sequence comprising e.g. sequences involved intranslation initiation, a (protein) coding region (cDNA or genomic DNA)and a 3′ non-translated sequence comprising e.g. transcriptiontermination sequence sites. A “chimeric gene” (or recombinant gene)refers to any gene, which is not normally found in nature in a species,in particular a gene in which one or more parts of the nucleic acidsequence are present that are not associated with each other in nature.For example the promoter is not associated in nature with part or all ofthe transcribed region or with another regulatory region. The term“chimeric gene” is understood to include expression constructs in whicha promoter or transcription regulatory sequence is operably linked toone or more coding sequences or to an antisense (reverse complement ofthe sense strand) or inverted repeat sequence (sense and antisense,whereby the RNA transcript forms double stranded RNA upontranscription).

“Identity” is a measure of the identity of nucleotide sequences or aminoacid sequences. In general, the sequences are aligned so that thehighest order match is obtained. “Identity” per se has an art-recognizedmeaning and can be calculated using published techniques. See, e.g.:(COMPUTATIONAL MOLECULAR BIOLOGY, Lesk, A. M., ed., Oxford UniversityPress, New York, 1988; BIOCOMPUTING: INFORMATICS AND GENOME PROJECTS,Smith, D. W., ed., Academic Press, New York, 1993; COMPUTER ANALYSIS OFSEQUENCE DATA, PART I, Griffin, A. M., and Griffin, H. G., eds., HumanaPress, New Jersey, 1994; SEQUENCE ANALYSIS IN MOLECULAR BIOLOGY, vonHeinje, G., Academic Press, 1987; and SEQUENCE ANALYSIS PRIMER;Gribskov, M. and Devereux, J., eds., M Stockton Press, New York, 1991).While a number of methods exist to measure identity between twopolynucleotide or polypeptide sequences, the term “identity” is wellknown to skilled artisans (Carillo, H., and Lipton, D., SIAM J. AppliedMath (1988) 48:1073). Methods commonly employed to determine identity orsimilarity between two sequences include, but are not limited to, thosedisclosed in GUIDE TO HUGE COMPUTERS, Martin J. Bishop, ed., AcademicPress, San Diego, 1994, and Carillo, H., and Lipton, D., SIAM J. AppliedMath (1988) 48:1073. Methods to determine identity and similarity arecodified in computer programs. Preferred computer program methods todetermine identity and similarity between two sequences include, but arenot limited to, GCS program package (Devereux, J., et al., Nucleic AcidsResearch (1984) 12(1):387), BLASTP, BLASTN, FASTA (Atschul, S. F. etal., J. Molec. Biol. (1990) 215:403).

As an illustration, by a polynucleotide having a nucleotide sequencehaving at least, for example, 95% “identity” to a reference nucleotidesequence encoding a polypeptide of a certain sequence it is intendedthat the nucleotide sequence of the polynucleotide is identical to thereference sequence except that the polynucleotide sequence may includeup to five point mutations per each 100 nucleotides of the referencepolypeptide sequence. In other words, to obtain a polynucleotide havinga nucleotide sequence at least 95% identical to a reference nucleotidesequence, up to 5% of the nucleotides in the reference sequence may bedeleted and/or substituted with another nucleotide, and/or a number ofnucleotides up to 5% of the total nucleotides in the reference sequencemay be inserted into the reference sequence. These mutations of thereference sequence may occur at the 5′ or 3′ terminal positions of thereference nucleotide sequence, or anywhere between those terminalpositions, interspersed either individually among nucleotides in thereference sequence or in one or more contiguous groups within thereference sequence.

Similarly, by a polypeptide having an amino acid sequence having atleast, for example, 95% “identity” to a reference amino acid sequence ofSEQ ID NO: 1 is intended that the amino acid sequence of the polypeptideis identical to the reference sequence except that the polypeptidesequence may include up to five amino acid alterations per each 100amino acids of the reference amino acid of SEQ ID NO: 1. In other words,to obtain a polypeptide having an amino acid sequence at least 95%identical to a reference amino acid sequence, up to 5% of the amino acidresidues in the reference sequence may be deleted or substituted withanother amino acid, or a number of amino acids up to 5% of the totalamino acid residues in the reference sequence may be inserted into thereference sequence. These alterations of the reference sequence mayoccur at the amino or carboxy terminal positions of the reference aminoacid sequence or anywhere between those terminal positions, interspersedeither individually among residues in the reference sequence or in oneor more contiguous groups within the reference sequence.

A nucleic acid according to the present invention may include anypolymer or oligomer of pyrimidine and purine bases, preferably cytosine,thymine, and uracil, and adenine and guanine, respectively (See AlbertL. Lehninger, Principles of Biochemistry, at 793-800 (Worth Pub. 1982)which is herein incorporated by reference in its entirety for allpurposes). The present invention contemplates any deoxyribonucleotide,ribonucleotide or peptide nucleic acid component, and any chemicalvariants thereof, such as methylated, hydroxymethylated or glycosylatedforms of these bases, and the like. The polymers or oligomers may beheterogenous or homogenous in composition, and may be isolated fromnaturally occurring sources or may be artificially or syntheticallyproduced. In addition, the nucleic acids may be DNA or RNA, or a mixturethereof, and may exist permanently or transitionally in single-strandedor double-stranded form, including homoduplex, heteroduplex, and hybridstates.

As used herein, the term “operably linked” refers to a linkage ofpolynucleotide elements in a functional relationship. A nucleic acid is“operably linked” when it is placed into a functional relationship withanother nucleic acid sequence. For instance, a promoter, or rather atranscription regulatory sequence, is operably linked to a codingsequence if it affects the transcription of the coding sequence.Operably linked means that the DNA sequences being linked are typicallycontiguous and, where necessary to join two protein encoding regions,contiguous and in reading frame so as to produce a “chimeric protein”. A“chimeric protein” or “hybrid protein” is a protein composed of variousprotein “domains” (or motifs) which is not found as such in nature butwhich a joined to form a functional protein, which displays thefunctionality of the joined domains. A chimeric protein may also be afusion protein of two or more proteins occurring in nature. The term“domain” as used herein means any part(s) or domain(s) of the proteinwith a specific structure or function that can be transferred to anotherprotein for providing a new hybrid protein with at least the functionalcharacteristic of the domain.

“Plant” refers to either the whole plant or to parts of a plant, such ascells, tissue or organs (e.g. pollen, seeds, gametes, roots, leaves,flowers, flower buds, anthers, fruit, etc.) obtainable from the plant,as well as derivatives of any of these and progeny derived from such aplant by selfing or crossing. “Plant cell(s)” include protoplasts,gametes, suspension cultures, microspores, pollen grains, etc., eitherin isolation or within a tissue, organ or organism.

As used herein, the term “promoter” refers to a nucleic acid fragmentthat functions to control the transcription of one or more genes,located upstream with respect to the direction of transcription of thetranscription initiation site of the gene, and is structurallyidentified by the presence of a binding site for DNA-dependent RNApolymerase, transcription initiation sites and any other DNA sequences,including, but not limited to transcription factor binding sites,repressor and activator protein binding sites, and any other sequencesof nucleotides known to one of skill in the art to act directly orindirectly to regulate the amount of transcription from the promoter.Optionally the term “promoter” includes herein also the 5′ UTR region(5′ Untranslated Region) (e.g. the promoter may herein include one ormore parts upstream (5′) of the translation initiation codon of a gene,as this region may have a role in regulating transcription and/ortranslation. A “constitutive” promoter is a promoter that is active inmost tissues under most physiological and developmental conditions. An“inducible” promoter is a promoter that is physiologically (e.g. byexternal application of certain compounds) or developmentally regulated.A “tissue specific” promoter is only active in specific types of tissuesor cells. A “promoter active in plants or plant cells” refers to thegeneral capability of the promoter to drive transcription within a plantor plant cell. It does not make any implications about thespatio-temporal activity of the promoter.

The terms “protein” or “polypeptide” are used interchangeably and referto molecules consisting of a chain of amino acids, without reference toa specific mode of action, size, 3 dimensional structure or origin. A“fragment” or “portion” of a protein may thus still be referred to as a“protein”. An “isolated protein” is used to refer to a protein which isno longer in its natural environment, for example in vitro or in arecombinant bacterial or plant host cell.

A “genetically modified plant” refers herein to a plant or plant cellhaving been transformed, e.g. by the introduction of an exogenous geneor additional copy or copies of an endogenous gene, said exogenous geneor additional endogenous gene may be integrated into the genome. Atransgenic plant cell transformed with an (isolated) polynucleotidesequence and plant cells and plants regenerated therefrom, are allunderstood to comprise said (isolated) polynucleotide sequence. Atransgenic plant cell may refer to a plant cell in isolation or intissue culture, or to a plant cell contained in a plant or in adifferentiated organ or tissue, and both possibilities are specificallyincluded herein. Hence, a reference to a plant cell in the descriptionor claims is not meant to refer only to isolated cells or protoplasts inculture, but refers to any plant cell, wherever it may be located or inwhatever type of plant tissue or organ it may be present. Methods forobtaining transgenic plant cells and plants are well known in the artand include but are not limited to Agrobacterium-mediated transformationof plant explants, particle bombardment of plant explants,transformation of plant explants using whiskers technology,transformation using viral vectors, electroporation of plantprotoplasts, direct uptake of DNA by protoplasts using polyethyleneglycol, microinjection of plant explants and/or protoplasts.Agrobacterium-mediated transformation is a preferred method to introducethe nucleic acid molecule of the invention into plant explants.Agrobacterium tumefaciens harbors a natural vector called Ti plasmidwhich was engineered to make it suitable for introduction of exogenousnucleic acid molecules into plant genomes. For genetic transformation,plant-derived explants are incubated with suspension of Agrobacteriumcells followed by cultivation of the explants on the medium containing aselective agent that promotes growth and regeneration of the transformedcells only.

As used herein, the “isolated plant drought-resistance protein(polypeptide)”, “isolated polypeptide that improves the plantdrought-resistant ability”, “isolated BccJAZ5a protein” or “isolatedBccJAZ5a polypeptide” refers to the BccJAZ5a protein substantially freeof the other proteins, lipids, saccharides and other substances that maybe naturally associated with said protein. A skilled person in the artcan utilize standard protein purification techniques to purify theBccJAZ5a protein. The substantially pure polypeptide forms a singlemajor band on a non-reduced polyacrylamide gel.

DETAILED DESCRIPTION OF THE INVENTION

The present inventors have isolated for the first time a new plantdrought-resistance gene from Brassica spp., which can be used to provideimproved drought-resistance or drought resistance in a plant. Theisolated gene is named as “BccJAZ5a”, based on which, transgenic plantswith improved drought-resistant ability can be produced.

The current invention relates to the improvement of drought resistanceof a plant by modifying the expression of a gene in said plant. Theimprovement is relative to a plant in which such modification has notbeen introduced or is not present. Such plant is preferably of the samespecies and/or variety. In other words, a modified plant according tothe invention is, in comparison to the non-modified plant, better ableto grow and survive under conditions of less water availability,water-deprivation or conditions of drought.

Drought stress, i.e. a period of limited availability of suitable wateras described above, may also manifest itself in a wilted state of theplant, i.e. in a state wherein a plant or part thereof has reducedturgor compared to a state wherein water is sufficiently available tothe plant. In this regard, a modified plant with enhanced expression(for example with enhanced, derepressed, or insertion of a gene) and/oractivity of the JAZ5a protein may be less wilted than a correspondingnon-modified plant if exposed to drought stress for the same time periodunder the same conditions.

Limited availability of water or drought is to be understood as asituation wherein water is or may become a limiting factor for biomassaccumulation or crop yield for a non-transformed or naturally occurringplant grown under such condition. For a plant obtained according to amethod according to the present invention and grown under saidcondition, water may not, or to a lesser degree, be a limiting factor.

It is believed by the current inventors that by enhancing expression(e.g. by enhancing, derepression or insertion of a gene) of JAZ5a leadsto the presence (or increased presence) of functional JAZ5a protein,either as the consequence of high expression or as the consequence ofincreased activity/functionality of the JAZ5a protein, or both, and thatsaid presence (or increased presence) of functional JAZ5a protein leadsto decreased need for water and/or improved resistance to drought ofsaid plant.

In one embodiment the use is provided of a protein for providing a plantwith drought resistance, wherein the protein is:

(a) a protein having the amino acid sequence of SEQ ID NO:4; or(b) a protein derived from the protein of (a) by substitution, deletionor addition of one or more residues in the amino acid sequence of SEQ IDNO:4 and wherein the protein is functionally equivalent to the aminoacid sequence represented by SEQ ID NO:4; or(c) a protein having at least 60% identity to the amino acid sequence ofSEQ ID NO:4 and having the same function as that of the amino acidsequence represented by SEQ ID NO:4.

In one embodiment, the said plant drought-resistance protein has atleast 60%, 65%, 70%, 75%, 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98% or 99%identity with the amino acid sequence represented by SEQ ID NO:4.

In one embodiment, the plant drought-resistance protein has 1-20,preferably 1-10, more preferable 1-5, most preferably 1-3 residuessubstituted, deleted or added in the amino acid sequence of SEQ ID No:4.

In one embodiment, the plant is a plant of Cruciferae. In oneembodiment, the Cruciferae plant is selected from the group consistingof Brassica spp. plant and Abrabidopsis spp. plant. In one embodiment,the Brassica spp. plant is Brassica campestris ssp. pekinensis.

In one embodiment, the Abrabidopsis spp. plant is Arabidopsis thaliana(L.) Heynh. In one embodiment, the plant drought-resistance protein isderived from the Brassica spp. Plant, preferably derived from Brassicacampestris L. ssp. chinensis.

In one embodiment, the use of a polynucleotide is provided for providinga plant with drought resistance, which is selected from the groupconsisting of:

(i) a polynucleotide encoding said protein; or(ii) a polynucleotide complementary to the polynucleotide of (i).In one embodiment, the nucleotide sequence of said polynucleotide is setforth in SEQ ID NO: 1 or 2. In an embodiment, a chimeric gene isprovided comprising such polynucleotide. In one embodiment, a vector isprovided, which contains said polynucleotide. Said vector may be chosenbased on the host cell or host plant used. One of ordinary skill in theart is capable of selecting a suitable vector for a specified host cell.

It is clear to the person skilled in the art that genes, including thepolynucleotides of the invention, can be cloned on basis of theavailable nucleotide sequence information, such as found in the attachedsequence listing, by methods known in the art. These include e.g. thedesign of DNA primers representing the flanking sequences of such geneof which one is generated in sense orientations and which initiatessynthesis of the sense strand and the other is created in reversecomplementary fashion and generates the antisense strand. Thermo stableDNA polymerases such as those used in polymerase chain reaction arecommonly used to carry out such experiments. Alternatively, DNAsequences representing genes can be chemically synthesized andsubsequently introduced in DNA vector molecules that can be multipliedby e.g. compatible bacteria such as e.g. E. coli.

In one embodiment, a genetically engineered host cell is provided, whichcomprises said vector or which comprises said polynucleotide integratedin the genome. In one embodiment, a plant is provided, which comprisesany of the aforementioned polynucleotides.

In one embodiment, a method for preparing the aforementioned protein isprovided, which comprises:

(a) culturing said host cell under conditions suitable for expression;(b) isolating said protein from the culture.

In one embodiment, the use is provided of the aforementioned protein ora polynucleotide encoding said protein for providing a plant with(improved) drought-resistance. In one embodiment, the use is forproviding a plant with (improved) drought-resistance in the boltingstage.

In one embodiment, a method for providing a plant with improved droughtresistance is provided, which comprises enhancing the expression oractivity of the aforementioned protein in said plant. In one embodiment,said method comprises transforming a polynucleotide encoding theaforementioned protein into the genome of the plant. In one embodiment,said method comprises:

(1) providing an Agrobacterium an expression vector comprising apolynucleotide encoding the protein of the invention;(2) providing a plant cell, organ or tissue;(3) contacting the plant cell, organ or tissue of step (2) with theAgrobacterium of step (1), such that the polynucleotide encoding theprotein of the invention is introduced into the plant cell.(3) optionally, selecting the plant cell, organ or tissue into which thepolynucleotide encoding the protein of the invention was introduced;(4) regenerating the plant cell, organ or tissue of step (3) into aplant.In one embodiment, the polynucleotide encoding the protein of theinvention is integrated into the chromosome of the plant cell.

In one embodiment a genetically modified plant is provided comprising apolynucleotide encoding the plant drought-resistance protein of theinvention.

In one embodiment of the present invention, a molecular marker foridentifying drought-resistance in a plant is provided, wherein saidmolecular marker comprises at least 30, 35, 40, 45, 50, or more(contiguous) nucleotides of the sequence of SEQ ID. No 1 or 2. In oneembodiment, a method is provided for identifying such molecular marker,said method comprising the step of sequencing the DNA of a plant cell.In one embodiment, a method for identifying such molecular marker isprovided comprising the step of amplifying the said sequence of SEQ IDNo. 1 or 2 and detecting the amplicon. In one embodiment, a pair ofprimers is provided capable of amplifying the said sequence of SEQ IDNo. 1 or 2, in a further embodiment, the pair of primers provided isrepresented by the nucleotide sequences SEQ ID NO: 5 and 6.

There is no specific limitation on the plants that can be used in thepresent invention, as long as the plant can be transformed, e.g. using agene, chimeric gene or vector. The plants include various crops, flowerplants or plants of forestry, etc. Specifically, the plants include, butare not limited to, dicotyledon, monocotyledon or gymnosperm. Morespecifically, the plants include, but is not limited to, wheat, barley,rye, rice, corn, sorghum, beet, apple, pear, plum, peach, apricot,cherry, strawberry, Rubus swinhoei Hance, blackberry, bean, lentil, pea,soy, rape, mustard, opium poppy, olea europea, helianthus, coconut,plant producing castor oil, cacao, peanut, calabash, cucumber,watermelon, cotton, flax, cannabis, jute, citrus, lemon, grapefruit,spinach, lettuce, asparagus, cabbage, Brassica campestris L. ssp.Pekinensis, Brassica campestris L. ssp. chinensis, carrot, onion,murphy, tomato, green pepper, avocado, cassia, camphor, tobacco, nut,coffee, aubergine, sugar cane, tea, pepper, grapevine, nettle grass,banana, natural rubber tree and ornamental plant, etc.

The term “plant(s)” includes, but is not limited to, plants ofCruciferae, Gramineae and Rosaceae. For example, the “plant” includesbut is not limited to Brassica campestris L. ssp. Pekinensis andBrassica campestris L. ssp. chinensis of Brassica spp. of theCruciferae; Abrabidopsis spp. plant of the Cruciferae; rice ofGramineae; and tobacco, melon and fruit, vegetable, rape and the like.More preferably, the “plant” is a plant of the Brassica spp. orAbrabidopsis spp. of the Cruciferae.

The polypeptide of the present invention can be a recombinantpolypeptide, a natural polypeptide or a synthetic polypeptide.Preferably, it is a recombinant polypeptide. The polypeptide of thepresent invention can be a product purified from a natural source,chemically synthesized, or recombinantly produced by prokaryotic oreukaryotic hosts (such as, bacterium, yeast, higher plant, insect andmammalian cell). According to the host used in the recombinantproduction, the polypeptide of the present invention can be glycosylatedor non-glycosylated. The polypeptide of the current invention canfurther include or not include the first native methionine residue.

The present invention further includes fragments, derivatives andanalogs of the BccJAZ5a protein. As used herein, the terms “fragment”,“derivative” and “analog” refer to the polypeptide that havesubstantively the same biological function and/or activity of theBccJAZ5a protein of the present invention. The polypeptide fragment,derivative or analog of the present invention may be (i) a polypeptidein which one or several conservative (preferred) or non-conservativeamino acid residues are substituted by one or more amino acid residuesthat are genetically encoded or not, or (ii) a polypeptide with one ormore amino acid residues bearing a substituent, or (iii) a fusionpolypeptide of the mature polypeptide and another compound (such as acompound for extending the half life of the polypeptide, such aspolyethylene glycol), or (iv) a polypeptide formed by an additionalamino acid sequence (such as a leader sequence or a secretion sequence,or a sequence facilitating purification, or a proteinogen sequence, or afusion protein) fusing to the polypeptide sequence. According to thedefinitions provided herein, these fragments, derivatives and analogscan be understood by the skilled in the art.

As used herein, the term “BccJAZ5a protein” refers to a polypeptideproviding improved drought-resistance or drought-resistance to plantsbased on the sequence of SEQ ID NO:4. This term also includes variantsof SEQ ID NO:4 that provide (improved) plant drought-resistance ability.Mutations include but are not limited to deletion, insertion and/orsubstitution of one or more (generally 1-50, preferably 1-30, morepreferably 1-20, most preferably 1-10, further more preferably 1-8 or1-5) amino acids, and addition or deletion of one or more (generallywithin 20, preferably within 10, more preferably within 5) amino acidsat the C-terminus and/or N-terminus. For example, it is understood thatsubstitution with an amino acid residue having close or similar propertywill generally not affect the function of the protein. Further, forexample, addition or deletion of one or more amino acids from theC-terminus and/or N-terminus will generally not affect the function ofthe protein. The term also includes the active fragments and activederivatives of the BccJAZ5a protein.

Variants of the polypeptide include its homologous sequences,conservative mutants, allelic mutant, natural mutant, induced mutant,protein encoded by a DNA that could hybridize to the DNA of BccJAZ5aprotein under a high or low stringent condition, and polypeptide orprotein obtained by utilizing an anti-serum against the BccJAZ5aprotein. The present invention also provides more related polypeptides,such as fusion proteins containing BccJAZ5a protein or fragmentsthereof. In addition to the full-length or almost full-lengthpolypeptides, the present invention also includes the soluble fragmentsof the BccJAZ5a protein. Generally, the fragment contains at least about20, generally at least about 30, preferably at least about 50, morepreferably at least about 80, most preferably at least about 100continuous amino acid of the BccJAZ5a protein.

The present invention also provides analogs of the BccJAZ5a protein orpolypeptide. These analogs may be different from the native BccJAZ5aprotein in the primary sequence or in modification patters along thesame primary sequence, or both. These polypeptides include the naturalor induced genetic mutants. The induced mutants may be obtained viavarious techniques, for example, by radiation or by exposure to amutagen so as to produce a random mutagenesis. They may also be obtainedby site-directed mutagenesis or some other known biologicaltechnologies. The analogs also include those having residues differentfrom the natural L-amino acid (such as D-amino acid), and those havingun-natural or synthetic amino acid(s), such as β- and γ-amino acids. Itshould be understood that the polypeptide of the subject invention isnot limited to the above representative examples.

Modification patterns, which will not change the primary structure,include in vivo or in vitro chemical derivation, such as acetylation orcarboxylation. Modification may also be glycosylation. Modification mayalso be phosphorylation of the amino acid residues (such as,phosphorylated tyrosine, phosphorylated serine, and phosphorylatedthreonine) in the sequence. Also included are polypeptides which aremodified to have an improved anti-proteolysis property or optimize thesolubility property.

In the present invention, “a conservative mutant of BccJAZ5a protein”refers to a polypeptide having up to 20, preferably up to 10, morepreferably up to 5, most preferably up to 3 amino acids in the aminoacid sequence of SEQ ID NO:4 being replaced by the amino acids withsimilar or close property. These mutant polypeptides preferably areproduced according to the amino acid replacement shown below in Table 1.

TABLE 1 Amino acid residue Representative substitution Preferredsubstitution Ala (A) Val; Leu; Ile Val Arg (R) Lys; Gln; Asn Lys Asn (N)Gln; His; Lys; Arg Gln Asp (D) Glu Glu Cys (C) Ser Ser Gln (Q) Asn AsnGlu (E) Asp Asp Gly (G) Pro; Ala Ala His (H) Asn; Gln; Lys; Arg Arg Ile(I) Leu; Val; Met; Ala; Phe Leu Leu (L) Ile; Val; Met; Ala; Phe Ile Lys(K) Arg; Gln; Asn Arg Met (M) Leu; Phe; Ile Leu Phe (F) Leu; Val; Ile;Ala; Tyr Leu Pro (P) Ala Ala Ser (S) Thr Thr Thr (T) Ser Ser Trp (W)Tyr; Phe Tyr Tyr (Y) Trp; Phe; Thr; Ser Phe Val (V) Ile; Leu; Met; Phe;Ala Leu

The present invention further provides polynucleotide sequences encodingthe BccJAZ5a protein of the current invention or variant polypeptidesthereof.

The polynucleotides of the present invention may be DNA or RNAmolecules. The DNA molecules include cDNA, genomic DNA and syntheticDNA. The DNA molecules may be in the form of a single strand or ofdouble strands. The DNA molecule may be the coding strand or thenon-coding strand. The coding sequence encoding the mature polypeptidemay be identical to the coding sequence of SEQ ID NO: 1 or 2, or may betheir degeneration variants. As used therein, “a degeneration variant”refers to a nucleic acid molecule that encodes a protein having thesequence of SEQ ID NO: 4 with a nucleotide sequence different from thecoding sequence as set forth in SEQ ID NO: 1 or 2.

The polynucleotides encoding polypeptide of SEQ ID NO:4 may comprise acoding sequence only encoding the mature polypeptide; a coding sequenceof the mature polypeptide and an additional coding sequence; the codingsequence of the mature polypeptide and a non-coding sequence, optionallyas well as an additional coding sequence.

The term “polynucleotide encoding a polypeptide” may optionally include,in addition to the polynucleotide encoding said polypeptide, anadditional coding and/or a non-coding polynucleotide.

The present invention further relates to variants of the abovepolynucleotides, which encode the same amino acid sequence of thepolypeptide of the present invention, and fragments, analogs andderivatives thereof. The variants of the polynucleotides may be thenaturally occurring allelic mutants or non-naturally occurring mutants.The nucleotide variants include substitution variants, deletion variantsand insertion variants. As known in the prior art, an allelic variant isan alternative form of a polynucleotide, wherein the mutation may besubstitution, deletion or insertion of one or more nucleotides, but thefunction of the polypeptide encoded by the allelic variant issubstantively un-altered.

The present invention also relates to a polynucleotide hybridizing toany of the above sequences and having at least 50%, preferably at least70%, more preferably at least 80% sequence identity between the twosequences. The present invention specifically relates to apolynucleotide hybridizing to the polynucleotides of the presentinvention under stringent conditions. In the present invention, the“stringent condition” refers to: (1) hybridization and elution at arelatively lower ionic strength and relatively higher temperature, suchas 0.2×SSC, 0.1% SDS, 60° C.; or (2) presence of denaturation agentduring hybridization, such s 50% (v/v) formamide, 0.1% calf serum/0.1%Ficoll, 42° C., and the like; or (3) conditions only allowinghybridization between two sequences that have at least 80%, preferablyat least 90%, more preferably at least 95% identity. Moreover, thepolypeptide encoded by the hybridizing polynucleotide exhibits the samebiological function and activity as those of the mature polypeptide asshown in SEQ ID NO: 4.

The present invention also relates to nucleic acid fragments that canhybridize to the any of the above sequences. As used herein, a “nucleicacid fragment” contains at least 15 nucleotides, preferably at least 30nucleotides, more preferably at least 50 nucleotides, most preferably atleast 100 nucleotides. The fragment of nucleic acid may be used in theamplification technique of nucleic acid (such as PCR) to determineand/or isolate the polynucleotide encoding the BccJAZ5a protein.

The full-length nucleotide sequence of the BccJAZ5a protein of thepresent invention or fragment thereof can typically be prepared via PCRamplification method, recombinant method or artificial synthesis. As toPCR amplification, the sequences of interests can be amplified bydesigning primers according to the related nucleotide sequence disclosedin the present invention, e.g. the open-reading frame, and using acommercially available cDNA library or a cDNA library prepared accordingto any of the conventional methods known in the art as a template. For alarge sequence, two or more PCR amplications may be needed, thefragments obtained in each amplification may be fused together, e.g. vialigation, in a correct orientation.

Once the sequence is obtained, it can be produced in a large amountusing recombinant techniques. The sequence may be cloned into a vector.The vector can be transformed into a cell, and then the sequence can beisolated from proliferated host cells using conventional means.

Furthermore, the related sequence can be synthesized by artificialsynthesis, especially when the fragment is relatively short. Generally,several small fragments are first synthesized and then fuseg, e.g. vialigation or fusion PCR, into a large fragment. The DNA sequence encodingthe protein (or fragment, or variant, or derivative thereof) of thepresent invention can be prepared via chemical synthesis. The obtainedDNA sequence can be incorporated into various known DNA molecules (suchas vectors) and then into cells. Further, mutations can be introducedinto the protein sequence (i.e. the sequence encoding the proteinsequence) of the present invention through the chemical synthesis.

The present invention also relates to a vector comprising thepolynucleotide of the present invention, a host cell geneticallyengineered to comprise the vector or the coding sequence of the BccJAZ5aprotein of the present invention, and a method for recombinantlyproducing the polypeptide of the present invention.

The polynucleotide of the present invention can be used to express orproduce a recombinant BccJAZ5a protein using conventional recombinantDNA techniques. The following steps may be included in such a use:

(1) Transforming or transfecting a host cell with a polynucleotide (orits variant) encoding the BccJAZ5a protein of the present invention, ora recombinant expression vector comprising said polynucleotide;(2) culturing the host cell in a culture medium;(3) isolating and purifying the protein from the culture medium or thecultured cells.

In the present invention, the polynucleotide sequence of the BccJAZ5aprotein can be inserted into a recombinant expression vector. The term“recombinant expression vector” refers to a bacterial plasmid, phage,yeast plasmid, plant cell virus, mammalian cell virus and any othervectors known in the art. In summary, any plasmids and vectors can beused as long as they can replicate and retain stably in the host.Expression vectors can contain a replication origin, promoter, markersand translation control element.

Various methods known in the art can be used to construct an expressionvector containing a DNA sequence encoding the BccJAZ5a protein andtranscription/translation regulatory signals. These methods include invitro recombinant techniques, DNA synthesis, in vivo recombinanttechniques, etc. The DNA sequence may be operably linked under apromoter for directing mRNA synthesis in the expression vector. Theexpression vector can further include a ribosome binding site forinitiating the translation and a transcription terminator.

Further, the expression vector can contain one or more selectivelylabeled genes to provide phenotypic traits for selecting the transformedhost cells. The labeled genes may encode, for example, dihydrofolatereductase, neomycin resistance and green fluorescent protein (GFP) forculture of eukaryotic cells, and kanamycin or ampicillin resistance forE. coli. The vector comprising the above DNA sequence and suitablepromoter or regulatory sequence can be used to transform host cells forprotein expression.

The host cell may be a prokaryotic cell, such as bacterial cell; orlower eukaryotic cell, such as yeast cell; or higher eukaryotic cell,such as plant cell. Examples include E. coli, Streptomyces,Agrobacterium, fungi cell such as yeast, and plant cell, etc.

When expressing the polynucleotide of the present invention in a highereukaryotic cell, the transcription can be enhanced when an enhancersequence is inserted into the vector. The enhancer may be a cis-actingfactor of DNA, which may contain about 10 to 300 bp and acts on apromoter to enhance the transcription of the gene.

The person skilled in the art knows how to select a vector, promoter,enhancer and host cell.

Transformation of a host cell with the recombinant DNA can be carriedout using conventional techniques known by the person skilled in theart. When the hosts are prokaryotic cells, such as E. coli, thecompetent cells that can uptake the DNA may be harvested after theexponential growth phase and then treated by CaCl₂ method, accordingmethods known in the art. Another method is to use MgCl₂. If desired,the transformation could be conducted using electroporation. When thehost cell is of an eukaryotic origin, one or more of the following DNAtransfecting methods may be used: calcium phosphate precipitation,conventional mechanical method such as micro-injection, electroporation,liposome packing, etc. Transformation of plant may also be achieved byusing agrobacterium or gene gun transformation, and the like, such asleaf discs transformation, rice immature embryo transformation, etc. Thetransformed plant cell, tissue or organ can be regenerated into a plantvia conventional methods, so as to obtain a plant having altered traits.

The transformant may be cultured in conventional ways to express thepolypeptide encoded by the gene of the present invention. Depending onthe host cell used, the culture medium used in the culture may beselected from various (conventional) culture mediums. Culturing can becarried out under conditions suitable for growth of the host cell. Whenthe host cell grows to a suitable density, the selected promoter may beinduced by a suitable method (e.g. a temperature change or chemicalinduction), after which the cell may be further cultured for a period oftime.

In the above methods, the recombinant polypeptide can be expressed inthe cell, or on the cell membrane, or be secreted outside the cell. Ifdesired, the recombinant protein could be isolated and purified viavarious isolation methods by utilizing the physical, chemical or otherproperties of the protein. These methods are well known in the art.Examples include but are not limited to the conventional renaturationtreatment, treatment with protein precipitant (such as salting out),centrifugation, osmosis (for disrupting the bacterium), ultra-treatment,ultra-centrifugation, molecular sieve chromatography (gel filtration),adsorption chromatography, ion-exchange chromatography, liquidchromatography such as high performance liquid chromatography (HPLC) andthe other, and combinations thereof.

The recombinant BccJAZ5a can be used in many applications. For example,it can be used to screen for the antibody, polypeptide or the otherligands agonistic or antagonistic to the function of the BccJAZ5aprotein. Screening a polypeptide library with the expressed recombinantBccJAZ5a protein may help finding valuable polypeptide molecules thatcould inhibit or stimulate the function of the BccJAZ5a protein.

The whole polynucleotide of the present invention or a portion thereofcan be used as a probe, which may be fixed onto a microarray or a DNAchip (also termed as “gene chip”) to perform an analysis of genedifferential expression. Primers specific for the BccJAZ5a protein toperform RNA reverse transcription polymerase chain reaction (RT-PCR) forin vitro amplification can also be used to detect the transcriptionproducts of the BccJAZ5a protein.

The present invention also relates to a method for modifying a plant (toprovide improved drought-resistance or drought resistance to the plant),comprising enhancing or providing the expression of the BccJAZ5a gene orthe activity of encoded protein in the plant.

Methods for enhancing or providing the expression of the BccJAZ5a geneare well known in the art. For example, plants can be transformed withan expression construct carrying the BccJAZ5a coding gene to express theBccJAZ5a gene. A promoter can be used to enhance the expression of theBccJAZ5a gene. An enhancer (e.g. the first intron of the rice waxy geneor the first intron of the Actin gene, and the like) can be used toenhance the expression of the BccJAZ5a gene. Promoters that may be usedin the current invention include but is not limited to the 35S promoterand the Ubi promoter in rice and corn.

In one embodiment of the present invention, a method for obtaining aplant with enhanced expression of BccJAZ5a protein includes:

(1) providing an Agrobacterium strain comprising an expression vector,wherein the expression vector contains a polynucleotide encoding aBccJAZ5a protein;(2) contacting a plant cell, tissue or organ with the Agrobacterium ofstep (1) such that the polynucleotide encoding the BccJAZ5a protein istransferred into the plant cell and may be integrated into the genome,thereby transforming the plant cell;(3) optionally, selecting the plant cell or tissue transformed with thepolynucleotide encoding the BccJAZ5a protein; and(4) regenerating the plant cell or tissue of step (3) into a plant.

Any suitable conventional means, including reagents, temperature andpressure controls, can be used in this process.

The present invention also includes agonists to the BccJAZ5a protein ora polynucleotide encoding the BccJAZ5a protein of the invention. Sincethe agonists of the BccJAZ5a protein can regulate the activity orexpression of the BccJAZ5a protein, the said agonists can also providedrought-resistance or improvements thereof to a plant through affectingthe BccJAZ5a protein, to achieve improvements on traits.

The agonists of the BccJAZ5a protein may refer to any substance that canenhance the activity of BccJAZ5a, maintain the stability of BccJAZ5a,promote the expression of BccJAZ5a, prolong effect duration of BccJAZ5a,or promote transcription and translation of BccJAZ5a. These substancescan be used in the present invention as agents for enhancing thedrought-resistance of a plant.

In one embodiment of the present invention, a BccJAZ5a gene is provided,the genomic sequence of which is listed in SEQ ID NO: 1, and the cDNAsequence of which is indicated in SEQ ID NO: 2. Said gene encodes aprotein containing 270 amino acids (SEQ ID NO:4). Said BccJAZ5a geneprovides a new route for drought tolerance modification of plant.

The present invention will be further illustrated in combination withthe examples below. It should be understood that these examples are forillustrating the present invention, but not be understood to limit thescope of the present invention in any way. The experimental methods,wherein specific conditions are not indicated in the following examplesare performed using conventional conditions, such as those described inSambrook et al., Molecular Cloning: A Laboratory Manual (New York: ColdSpring Harbor Laboratory Press, 2002), or according to the conditionsrecommended by the manufacturer. Unless otherwise specificallyindicated, the percentage and part are calculated based on weight.Unless otherwise specifically indicated, all of the scientific termsused herein have the same meanings as those familiar to the skilled inthe art. Furthermore, any methods and materials equivalent to thedisclosed contents can be used in the present invention. The preferredpracticing method and material disclosed herein are just forillustrative purpose.

All references cited in the present invention are incorporated herein byreference as each one of them was individually cited. Further, it isunderstood that various modifications and/or changes are obvious to askilled person in the art, in view of above teaching of the currentinvention, falling within the scope as defined by the description andthe claims.

SEQUENCE LISTING

SEQ ID NO. 1: genomic DNA sequence encoding the BccJAZ5a proteinSEQ ID NO. 2: cDNA sequence encoding the BccJAZ5a proteinSEQ ID NO. 3: genomic DNA sequence encoding the BccJAZ5b proteinSEQ ID NO. 4: amino acid sequence of the BccJAZ5a proteinSEQ ID NO. 5: forward primer 5′ AAGAAGCCAAGTCTGTGA 3′SEQ ID NO. 6: reverse primer 5′ TCGGAGGATAATGATGAC 3′

Examples

Segregating T3 progeny (harvested from 6 individual plants) from twoindividual transformation events of Arabidopsis thaliana (At) with theBccJAZ5a encoding nucleotide sequence from Brassica rapa ssp. chinensisbehind a constitutive 35S promoter (35S::BccJAZ5a)(hereafter referred toas mutant seeds or mutant plants) were obtained from the NationalLaboratory of Plant Molecular Genetics, Shanghai Institute of PlantPhysiology and Ecology, Shanghai Institutes for Biological Sciences,Chinese Academy of Sciences, 300 Fenglin Road, Shanghai 200032, China.As control At Col-0 (Columbia, N60000; hereafter referred to as controlseed or plant) were obtained from the Nottingham Arabidopsis StockCentre (NASC; School of Biosciences, University of Nottingham, SuttonBonington Campus, Loughborough, LE12 5RD United Kingdom).

KeyGene N.V. and the Shanghai Institutes for Biological Sciences areparties to a joint research agreement.

For the gene “BccJAZ5” of the present invention two genomic sequencesare provided, which respectively are BccJAZ5a (copy a) and BccJAZ5b(copy b). The genomic sequence of BccJAZ5a is indicated in SEQ ID NO:1,its CDS sequence is indicated in SEQ ID NO:2. It encodes a protein“BccJAZ5a” having 270aa (SEQ ID NO:4). The genomic sequence of BccJAZ5bis shown in SEQ ID NO:3.

The expression of BccJAZa from Brassica rapa ssp. chinensis wasconfirmed with a semi-quantitative RT-PCR. Primers used were:

BccJAZ5a:

(SEQ ID NO: 5) Forward: 5′ AAGAAGCCAAGTCTGTGA 3′; (SEQ ID NO: 6)Reverse: 5′ TCGGAGGATAATGATGAC 3′.

Growth Medium:

A soil mixture comprising one part of sand, one part of vermiculite andtwo parts of compost was used (sand:vermiculite:compost=1:1:2). Thismixture increases the water percolation hence facilitates uniform wateruptake by each pot and better water drainage. Before sowing, the seedswere kept at 4° C. for 3 days under dark and humid conditions forstratification.

Both mutant and control seeds were sown in 60-ml baskets (ARABASKETS,Lehle seeds) with a density of 1 plant per pot. For each genotype, therewere 20 replicates. Baskets were kept in a rectangular tray containing8×5=40 holes of ˜4 cm diameter. Plants were cultivated in a growthchamber with a 16-hrs day (24° C.) and 8-hrs night (20° C.) at 60-70%relative humidity. The first 3 days after sowing the plants were kept at100% humidity by covering them with transparent lids or transparentplastic bags. Nutrient solution (EC=1.5) was supplied to all the plantsfrom the bottom of the pots in the tray 10 days after germination (DAG),and at 15 DAG the plants were subjected to drought (for 19, 20, or 21days) by transferring the pots to dry trays. Subsequently, plants wererehydrated and observed for recovery after 1 week.

Replicates of individually harvested mutant T3 progeny from twoindividual transformation events and replicates of control plants wereincluded. Total time needed for a complete test was approx. 36-39 days.

Drought Assay Examination

At 10 DAG, plants received nutrition (EC-1.5) and at 15 DAG each pot wasmoved to a dry tray. From this day onwards the plants did not receiveany water. While water was withheld pots were shuffled daily within thetrays to reduce the position effects and allow uniform evaporation.Every day the plants, especially the control (or wild type) (Col-0) wereobserved for wilting signs (see FIG. 1). On the 19^(th) day of drought(DOD), Col-0 wilted completely and did not recover upon rehydration. Wedetermined this day as its permanent wilting point (PWP). From this dayonwards replicates from the mutant were rehydrated and observed forrecovery signs and pictures were taken (see FIG. 2). Mutant plantsshowed survival for at least 1 day longer under drought than did thecontrol.

TABLE 2 Survival after rehydration. Batches of plants were rehydratedfrom 19 DOD onwards. Representative replicas were chosen to be actuallyrehydrated. Column 1^(st), 2^(nd), 3^(rd) DOR (day of rehydration)contains the data that indicates the ratio of plants that survived andthe plants that died. 1 DOR 2 DOR 3 DOR Line (19 DOD) (20 DOD (21 DOD)35S:BcPJAZ5a T3 seeds line 1, 1:1 2:2 0:4 #12 35S:BcPJAZ5a T3 seeds line1, 1:1 1:3 0:4 #13 Col-0 0:2 0:4 0:4 35S:BcPJAZ5a T3 seeds line 3, 2:02:2 0:4 #12 35S:BcPJAZ5a T3 seeds line 3, 2:0 1:3 0:4 #13 35S:BcPJAZ5aT3 seeds line 3, 2:0 3:1 1:3 #14 Col-0 1:1 0:4 0:4

Study on the Domains in the JAZ5a Protein, its Variants and Functions

The inventors of the subject application have identified domains in theBccJAZ5a protein (SEQ ID NO:4). Positions 101-130 constitute a tifydomain, and the segment of 184-209 is a CCT_2 motif. These domains maybe important active sites for the protein's drought-resistance function.

Based on the above analysis, the inventors construct several variants ofthe BccJAZ5a protein as specified below:

In the sequence of the BccJAZ5a protein (SEQ ID NO:4), amino acid 9 ischanged from A to V, so as to obtain BccJAZ5a-M1 variant.In the sequence of the BccJAZ5a protein (SEQ ID NO:4), amino acid 253 ischanged from L to I, so as to obtain BccJAZ5a-M2 variant.In the sequence of the BccJAZ5a protein (SEQ ID NO:4), amino acid 147 ischanged from V to A, so as to obtain BccJAZ5a-M3 variant, and amino acid230 is changed from L to I.In the sequence of the BccJAZ5a protein (SEQ ID NO:4), amino acids266-270 are deleted, so as to obtain BccJAZ5a-M4 variant.In the sequence of the BccJAZ5a protein (SEQ ID NO:4), amino acids159-161 are deleted, so as to obtain BccJAZ5a-M5 variant.In the sequence of the BccJAZ5a protein (SEQ ID NO:4), four amino acidsATAA are added to the C-terminus, so as to obtain BccJAZ5a-M6 variant.

The CDS sequence of the BccJAZ5a gene shown in SEQ ID NO: 2 is firstcloned into the pCAMBIA1300 vector at the Kpn I site to obtain arecombinant vector containing said CDS. Then, site-directed mutagenesisis conducted to introduce the corresponding substitution, deletion andaddition to obtain the recombinant vectors containing the above-saidvariants respectively.

The recombinant vectors thus constructed are transformed into strains ofagrobacterium, and then the agrobacterium strains are used to transformArabidopsis, so that transgenic Arabidopsis plants are obtained. Adrought treatment such as described above is used to verify thephenotype of these transgenic Arabidopsis plants. The transgenic plantsshow improved drought resistance as compared the wild type plants.

1. A method for selecting a plant with improved drought resistance,comprising: introducing into a plurality of plants or enhancingexpression or activity in the plants of a polynucleotide encoding adrought resistance protein of plant origin having the amino acidsequence of SEQ ID NO:4 or at least 95% sequence identity to the aminoacid sequence of SEQ ID NO:4, and measuring drought resistance of theplants to select a plant with improved drought resistance.
 2. The methodaccording to claim 1, wherein the plant is not Arabidopsis thaliana. 3.The method according to claim 1, wherein the step of introducing intothe plant or enhancing expression or activity in the plant of thedrought-resistance protein comprises transforming said plant with apolynucleotide encoding the drought-resistance protein.
 4. The methodaccording to claim 3, wherein the polynucleotide is incorporated intothe genome of the plant.
 5. The method according to claim 3, comprising:(1) providing an Agrobacterium strain containing an expression vectorcomprising a polynucleotide encoding the drought-resistance protein; (2)providing a plant cell, organ or tissue; (3) contacting the plant cell,organ or tissue of step (2) with the Agrobacterium strain of step (1)such that the polynucleotide encoding the drought-resistance protein isintroduced into the plant cell, organ or tissue; (4) optionally,selecting a plant cell; (5) growing the plant cell, organ or tissue intoa plant.
 6. The method according to claim 5, wherein after introductionof the polynucleotide in the plant cell, organ or tissue, thepolynucleotide integrates in the genome of the plant cell, organ ortissue.
 7. The method according to claim 1, wherein the plant isselected from the group consisting of dicotyledon, monocotyledon andgymnosperm.
 8. The method according to claim 1, wherein the plant isselected from the group consisting of wheat, barley, rye, rice, corn,sorghum, beet, apple, pear, plum, peach, apricot, cherry, strawberry,Rubus swinhoei Hance, blackberry, bean, lentil, pea, soy, rape, mustard,opium poppy, olea europea, helianthus, coconut, plant producing castoroil, cacao, peanut, calabash, cucumber, watermelon, cotton, flax,cannabis, jute, citrus, lemon, grapefruit, spinach, lettuce, asparagus,cabbage, Brassica campestris L. ssp. Pekinensis, Brassica campestris L.ssp. chinensis, carrot, onion, murphy, tomato, green pepper, avocado,cassia, camphor, tobacco, nut, coffee, aubergine, sugar cane, tea,pepper, grapevine, nettle grass, banana, natural rubber tree andornamental plant.
 9. The method according to claim 1, wherein thedrought-resistance protein has the amino acid sequence of SEQ ID NO:4.10. The method according to claim 1, wherein the drought-resistanceprotein has at least 95% sequence identity to the amino acid sequence ofSEQ ID NO:4.